Reactive oxygen species (ROS) are emerging as critical second messengers in many signaling pathways related to health and disease. While much progress has been made in understanding the mechanisms by which ROS levels are regulated inside cells, less is known about the molecular signaling events that occur downstream of ROS generation. Protein phosphatases are perhaps the best-characterized ROS effectors, suggesting that crosstalk readily occurs between ROS- and phosphorylation-dependent pathways. This notion is supported by recent studies demonstrating that several protein kinases are also directly regulated by ROS modification. Indeed, the reversible oxidation of specific Cys residues in redox-sensitive kinases has been shown to influence their activity (either positively or negatively), subcellular localization, and protein-protein interactions. In mny cases, the modified Cys in the affected kinase is conserved among other members in the same kinase family. This raises the possibility that reversible oxidation may be a general means of regulating kinase function inside cells. Therefore, in Aim 1, we will examine the redox sensitivity of kinases containing conserved Cys residues at positions known to be oxidized in other representative family members. In Aim 2, we will explore the impact of oxidation on substrate selection by redox-sensitive kinases using functional HuProt protein microarrays composed of 19,000 unique human proteins (representing ~90% of the human proteome). Our hypothesis is that oxidation will lead to differential phosphorylation of proteins on the microarrays and may even shift the substrate preference of a given kinase such that distinct sets of substrates are targeted by the oxidized and reduced forms of the kinase. Not only will these studies offer unique insights into oxidation-induced changes in substrate specificity but, due to the large number of proteins on the HuProt microarrays, they also promise to dramatically expand the number of known substrates for each kinase in the reference state (i.e., under reducing conditions). We will build upon our findings in Aims 1 and 2 through targeted analysis of redox-sensitive kinases and their substrates following activation of physiologically-relevant redox signaling pathways in cultured cells.